Aberrant signaling of protein kinases has been implicated in a variety of cancers. A prototypical example of a cancer caused by dysregulated kinase activity is chronic myelogenous leukemia (CML). The reciprocal translocation t(9;22)(q34;q11) of the Abl1 gene on chromosome 9 with part of the Bcr gene on chromosome 22 leads to a fusion protein, Bcr-Abl. An important auto-inhibitory myristoylation site, typically present on wild-type c-Abl, is lost upon forming the fusion protein. Bcr-Abl therefore possesses constitutive kinase activity and is the primary driver for CML.
The development of protein kinase inhibitors for use in oncology was spurred by the success of imatinib, which was approved for the treatment of CML in 2001. In clinical trials, imatinib showed remarkable efficacy in treating CML patients, increasing the 5-year overall survival rate from about 30% (prior to any targeted therapy) to an unprecedented 90%. However, long term treatment with imatinib invariably led to the development of resistance, due to point mutations in its target protein, Bcr-Abl.
Second-generation Bcr-Abl inhibitors nilotinib, dasatinib, and bosutinib were initially approved to counter imatinib-resistant CML. Since then, nilotinib and dasatinib have been approved for front-line CML therapy, while bosutinib is currently approved only for salvage therapy. Each of the four approved Abl inhibitors orthosterically compete with ATP to form hydrogen bonds with the hinge region of c-Abl (i.e. the loop that connects the N- and C-lobes of the kinase). Additionally, the highly-dynamic, glycine-rich, phosphate-binding loop (P-loop) of the kinase makes extensive van der Waals contacts with the hinge-binding motifs of these inhibitors. Dasatinib and bosutinib are termed type-I inhibitors and bind to a conformation of the kinase that closely mimics nucleotide-binding. This conformation, consisting of the activation loop residue D381 (of the DFG motif) positioned for catalysis and the adjacent F382 residue buried in a hydrophobic pocket, is termed the DFG-in active conformation. On the other hand, imatinib and nilotinib, classified as type-II inhibitors, bind to c-Abl in a DFG-out inactive conformation. In this conformation, the inhibitors extend past a so-called ‘gatekeeper’ residue (T315) into the allosteric hydrophobic pocket normally occupied by F382. Access to the allosteric pocket is facilitated by a 180° flip in the DGF motif.
Point mutations in the kinase domain of c-Abl inhibit binding of imatinib, either through direct or allosteric mechanisms. Over 50 single-point mutations have been identified in various positions of the kinase. The P-loop residues (G250, Y253, E255), gatekeeper residue T315, and the M351 and F359 residues account for over 70% of all mutated residues. The second generation inhibitors are effective against many P-loop mutants of c-Abl, however, like imatinib, they are completely ineffective against the T315I gatekeeper mutation. This specific mutation is the single most common mutation observed and greatly enhances kinase activity. The exact mechanism by which the T315I mutation abrogates binding of first and generation inhibitors is still a debated question. However, it is believed to be, at least in part, due to the elimination of a critical H-bond between the inhibitor and the hydroxyl group of the T315 residue.
Ponatinib, a third-generation type-II inhibitor, is the only drug approved for the treatment of refractory CML caused by T315I Bcr-Abl. Its use, however, has been complicated by its serious vascular adverse events (AEs). In a phase I trial, after a median follow-up of 2.7 years, 48% of patients had experienced serious vascular AEs, loss of blood flow and severe narrowing of blood vessels in the extremities, heart, and brain requiring urgent surgical procedures to restore blood flow. Twenty-seven percent of patients developed arterial or venous thrombosis and occlusions, while heart failure, including fatalities, occurred in 8% of patients. These AEs were found to be both cumulative and dose-dependent resulting in ponatinib being pulled off the market for a short time before being reinstated with a black-box warning.
Imatinib set the bench mark for Bcr-Abl inhibitors not only in its efficacy, but also in its excellent safety profile. The clinical safety of imatinib has been attributed to its narrow spectrum of selectivity. While second generation inhibitors are more promiscuous and show limited AEs, their safety profiles are superior to that of ponatinib. The exact molecular mechanism for the vascular AEs in ponatinib-treated patients is currently unknown, however, it has been correlated with its broad spectrum of selectivity. Particularly, the ability of ponatinib to potently inhibit VEGFR 1-3 (receptors kinases involved in vasculogenesis and angiogenesis) has been hypothesized to cause vascular toxicity. Additionally, ponatinib inhibition of the FGFR kinases is believed to enhance such AEs.
The success of imatinib-therapy has paradoxically led to an increase in the number of patients with CML. Because patients with CML must continue kinase inhibitor therapy for the rest of their lives, an increasing number of these patients will eventually develop resistance to imatinib. Because the median age of patients with CML is >60 years, in whom cardiovascular disease is prevalent, the safety of novel inhibitors of imatinib-resistant CML is critical.